The goals of the proposed research are to determine the mechanisms that lead to the development of necrotizing enterocolitis (NEC), which is the leading cause of death and disability from gastrointestinal disease in premature infants, and to determine novel therapeutic strategies for this devastating disorder. To accomplish these goals, we now focus on the innate immune receptor Toll like receptor-4 (TLR4), which is known to be the receptor for lipopolysaccharide (LPS), and which in the first funding period, we have identified to play a critical role in the pathogenesis of this disease. We now seek to define the molecular and cellular mechanisms by which TLR4 activation leads to NEC. To do so, we generated mice that lack TLR4 specifically on the intestinal epithelium, which were found to be protected from NEC development as compared with wild-type mice. In seeking to understand the mechanisms involved, we now demonstrate that TLR4 exerts an important and previously unrecognized role in the regulation of normal intestinal epithelial differentiation. We also determined that TLR4 is expressed on the intestinal stem cells (ISC's), and that ISC proliferation - a key determinant of the ability of the intestinal mucosa to heal - is reduced in NEC. To define whether TLR4 activation on ISC's was required for the loss of ISC proliferation, we generated mice that lack TLR4 specifically on the ISC's, which showed intact proliferation after TLR4 activation compared with wild-type mice. We now hypothesize that inappropriate TLR4 signaling in the preterm infant leads to the development of NEC by impairing the activity and function of the intestinal stem cells. To test this hypothesize we propose the following three aims: Aim 1. To investigate the role of TLR4 activation in regulating intestinal epithelial differentiation in the pathogenesis of necrotizing enterocolitis. Aim 2. To determine the effects of inappropriate activation of TLR4 in the developing intestine on the induction of intestinal inflammation. Aim 3. To characterize the role of TLR4-mediated loss of intestinal stem cell proliferation in the pathogenesis of necrotizing enterocolitis, and to determine whether restoring intestinal stem cell proliferation can prevent or attenuate the severity of the disease. To accomplish these research goals, we have generated mice strains that lack or overexpress TLR4 in intestinal epithelium or that lack TLR4 on the ISC's, have developed techniques of intestinal stem cell isolation and culture and have successfully introduced TLR4 agonists into the developing mouse intestine in utero. These studies will make a significant conceptual advance in linking the innate immune system with intestinal development, will advance our understanding of NEC by explaining the susceptibility of the premature infant based on the role of TLR4 in the intestinal stem cells, and will evaluate novel anti-NEC therapies which seek to reverse the deleterious effects of TLR4 on the ability of the intestinal stem cells to repair the injured mucosa of the premature small intestine. PUBLIC HEALTH RELEVANCE: Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in premature infants, and for which there exists no effective cure. The current proposal seeks to understand the causes of necrotizing enterocolitis by focusing on the interaction between the immune system and the stem cells of the newborn intestine, and therefore to discover novel treatments for this devastating disease.